Phytic Acid and : Implications for Protein Utilization by Poultry

A. J. Cowieson,*1 T. Acamovic,*2 and M. R. Bedford†

*Avian Science Research Centre, Scottish Agricultural College, Ayr Campus, Ayr, KA6 5HW, UK; and †Zymetrics Inc., Chestnut House, Beckhampton, Marlborough, Wiltshire, SN8 1QJ, UK

ABSTRACT The effect of the ingestion of myo- acids, N, and DM of casein compared with birds fed hexaphosphate (IP6) and phytase (EC 3.1.3.26) on the di- casein alone. Supplementation of the mixture of casein < gestibility of casein was investigated using growing and IP6 with phytase improved (P 0.05) the digestibility broiler chickens. A total of 64 female Ross broilers were coefficients of amino acids compared with birds fed on used in a precision feeding study. One group of 8 birds casein and IP6 with no supplemental phytase. The excre- < was fed a solution of glucose to estimate endogenous tion of endogenous minerals was increased (P 0.05) < losses. Seven groups, each of 8 birds, were fed either by the ingestion of IP6 and reduced (P 0.05) by the casein, casein + 1,000 units of phytase activity (FTU), supplementation of IP6 with phytase. In the absence of exogenous phytase, the recovery of phytate-P in excreta casein + 2,000 FTU, casein + 0.5 g of IP , casein + 0.5 g 6 was approximately 80%. However, the recovery of phy- of IP + 1,000 FTU, casein+1gofIP, or casein +1gofIP 6 6 6 tate-P was significantly reduced by the addition of exoge- + 1,000 FTU. The excretion of DM, amino acids, nitrogen, nous phytase to the IP6/casein mixture. It can be con- minerals, and phytate- was determined over cluded that the ingestion of IP6 reduces the digestibility a 48-h period and digestibility coefficients were coefficients of amino acids and the metabolizability of calculated. Casein was found to be highly digestible, with nitrogen of casein. This is likely to be mediated partially true coefficients of DM, N, and amino acid digestibility through increased endogenous losses. However, the addi- of between 0.85 and 1.0. However, the ingestion of IP6 tion of phytase can partially ameliorate the detrimental < reduced (P 0.05) the digestibility coefficients of amino effects of IP6 on protein utilization. Key words: inositol , phytate, phytase, broilers, precision feeding 2006 Poultry Science 85:878–885

INTRODUCTION (Maenz et al., 1999; Maenz, 2001), forming insoluble salts at intestinal pH, reducing the availability of these miner- A variable but large proportion of the phosphorus (P) als for absorption (Lonnerdal et al., 1999; Selle et al., 2000; in material is in the form of phytate-P (myo-inositol Sandberg, 2002). Furthermore, the availability of dietary hexakis phosphate; Haug and Lantzsch, 1983; Eeckhout amino acids and energy is reduced with the presence of and De Paepe, 1994; Harland and Morris, 1995; Harland IP6 in the ration (Ravindran et al., 2000; Selle et al., 2000). and Narula, 1999; Sandberg, 2002). Phytate-P is largely Although exogenous phytase can improve the retention unavailable for utilization by poultry due to a lack of of dietary P, the addition of exogenous to poul- effective endogenous phytase, the responsible try diets improves performance parameters other than for the hydrolysis of phytate. Inositol hexaphosphate is those associated with an improvement in P utilization relatively reactive having 12 dissociable protons with pKa (Kemme et al., 1999; Ravindran et al., 2000, 2001; Selle et values that range from about 1.5 to 10 (Costello et al., al., 2000). The interaction between IP6 and protein is 1976). The reactivity of inositol phosphate is largely dependent on pH, with binary protein-IP6 com- highly dependent on the conformation and configuration plexes being formed at low pH and ternary protein-IP6- of the molecule and the pH of its environment (Costello complexes formed as pH increases toward neu- et al., 1976; Cheryan, 1980). Fully phosphorylated myo- trality (Selle et al., 2000; Maenz, 2001; Adeola and Sands, inositol (IP6) is a potent chelator of many mineral 2003). The complexation of minerals by phytate is likely to reduce their participation as cofactors in , es- pecially under suboptimal supplies. Knuckles et al. (1989) 2006 Poultry Science Association, Inc. demonstrated in vitro that esters of inositol phosphate Received July 22, 2005. inhibited the digestion of casein by pepsin, the extent of Accepted September 8, 2005. inhibition being a function of the degree of phosphoryla- 1Current address: Danisco Animal Nutrition, PO Box 777, Marlbor- ough, Wiltshire, SN8 1XN, UK. tion of the inositol ring. This inhibition may be a function 2Corresponding author: [email protected] of reduced enzyme activity or an interaction between

878 PHYTIC ACID, PHYTASE, AND PROTEIN UTILIZATION 879 inositol phosphate and protein. However, the interaction IP6, casein + 0.5 g of IP6 + 1,000 FTU, casein + 1 g of IP6, between amino acids and proteins, and IP6 in the gastroin- and casein + 1 g of IP6 + 1,000 FTU. Each treatment group testinal tract of poultry is not well understood and re- consisted of 8 replicate birds and a further 8 birds were quires further examination. fed a solution of glucose to estimate endogenous losses. The addition of exogenous phytases to the diets of After feeding, the birds were placed in individual metabo- poultry has been shown to improve weight gain, mineral lism cages and excreta were collected quantitatively over retention, ME, and amino acid digestibility (Ravindran a 48-h period. The total excretion of DM, N, amino acids, et al., 1999; Newkirk and Classen, 2001; Murai et al., 2002; phytate-P, and minerals were determined and, when ap- Rutherfurd et al., 2002; Augspurger et al., 2003; Cowieson propriate, digestibility coefficients were calculated. and Adeola, 2005). Phytases also reduce the excretion of phytate-P, particularly in diets that have been formulated Laboratory Analyses to have a low available P concentration (Zhang et al., 1999; Ahmad et al., 2000; Edens et al., 2000; Selle et al., Following collection, the excreta were freeze dried, 2000). The improvements in amino acid utilization associ- weighed, and milled to pass a 1-mm aperture. The total ated with the addition of exogenous phytases to poultry quantity of DM excreted was recorded and the content diets are likely to be mediated through both a reduced of N in the excreta was determined using a LECO FP- endogenous amino acid loss as well as an improvement 2000 Analyzer (LECO, St. Joseph, MI). Amino acid con- in the retention of dietary amino acids (Cowieson et al., centrations in the excreta were determined by HPLC fol- 2004). Previous work from this laboratory has demon- lowing acid hydrolysis. The HPLC system comprised a strated, in model systems using growing broiler chickens, Dionex ASI-100 autosampler fitted with a Dionex P580 that ingestion of IP increases the excretion of endogenous 6 pump and a Dionex RF-2000 detector (Sunnyvale, CA). compounds including amino acids (Cowieson et al., 2004). The flow rate was 1 mL/min and the column used was Because of the complexity of components in diets, it is a Spherisorb ODS2 (150 × 4.6 mm fitted with a Waters impossible to characterize with clarity what supplemental guard cartridge (Alltech Assoc., Camforth, UK). The min- dietary phytase affects to produce the improvements or eral content of the excreta, after digestion with nitric acid changes seen. The effects of both phytate and phytases in a microwave digestion unit, was determined using an can be studied in a model system in which variation is Optima 4300 DV Dual View ICP-OE spectrometer (Per- minimized. The purpose of the study reported here was to investigate the effects of supplemental exogenous phytase kin-Elmer, Beaconsfield, UK). The concentration of phy- tate-P in the excreta was determined colorimetrically us- and IP6 on the utilization of protein and amino acids from casein using a precision feeding procedure. ing the method of Haug and Lantzsch (1983).

MATERIALS AND METHODS Statistical Analysis and Digestibility Coefficient Calculation Bird Management Data were analyzed statistically using ANOVA (Gens- Sixty-four female Ross broilers were reared under stan- tat, release 5, IACR, Rothamstead, UK) to determine the dard commercial conditions until they reached the target main effects of IP6 and phytase. Tukey’s LSD was used BW of approximately 2.5 kg. During the growing phase, to detect significant differences between means. the birds were fed a standard broiler ration (calculated True digestibility coefficients of DM, N, amino acids, provision was 13.3 MJ of ME/kg and 210 g of CP/kg). and S were calculated using the formula [Nin − (Nexc − The precision feeding study was run according to the Nexcg)]/Nin, where Nin is the quantity of nutrient in- method of McNab and Blair (1988), as modified by Ferraz gested, Nexc is the quantity of nutrient excreted, and de Oliveira et al. (1994). Briefly, when the birds reached Nexcg is the mean quantity of nutrient excreted by the 8 the target weight (at approximately 42 d) they were birds fed the glucose solution. The apparent digestibility placed in a pen with a raised slatted floor and feed was coefficient of phytate-P was calculated using the formula withdrawn for a period of 48 h. Water was provided ad (pPin − pPexc)/pPin, where pPin is the quantity of phytate- libitum via suspended nipple drinker lines throughout P ingested and pPexc is the quantity of phytate-P excreted. the experimental period. After the 48-h period of feed deprivation, the birds were precision-fed a suspension of 5 g of casein in 50 mL of distilled water with and without Animal Ethics IP6 [aqueous solution of phytic acid (cat no. 593648), Sigma-Aldrich, Gillingham, UK] and phytase. The phy- The work was done with the approval of the Scottish tase (EC 3.1.3.26) used in this work was derived from Agricultural College Animal Experiments Committee. Escherichia coli and produced in Pichia pastoris (Zymetrics Inc., Minneapolis, MN), and was added based on guaran- RESULTS teed activities. Phytase activity was measured in phytase activity units (FTU). The treatments used were casein, There were no mortalities during the study and all casein + 1,000 FTU, casein + 2,000 FTU, casein + 0.5 g of birds appeared healthy throughout. 880 COWIESON ET AL.

Table 1. Chemical composition of casein (determined)1 with birds fed casein without IP6. There was an adverse Item effect of IP6 on the digestibility coefficients of alanine, serine, aspartic acid, and threonine compared with the DM, g/kg 890 N, g/kg 138 other amino acids. It should be noted however, that the Asp, g/16 g of N 7.6 ingestion of both 0.5 and1gofIP6 with the casein im- Ser, g/16 g of N 6.1 paired (P < 0.05) the digestibility of all amino acids com- Glu, g/16 g of N 25.8 pared with birds fed casein alone. The metabolizability Ala, g/16 g of N 3.2 < Tyr, g/16 g of N 5.9 of N was reduced (P 0.05) by the ingestion of IP6 to His, g/16 g of N 2.7 a greater extent than that observed for digestibility of Thr, g/16 g of N 4.6 Arg, g/16 g of N 3.8 amino acids. Val, g/16 g of N 7.3 Addition of phytase to the mixture of casein and 0.5 g < Phe, g/16 g of N 5.5 of IP6 increased (P 0.05) the digestibility coefficients of Ile, g/16 g of N 5.5 > Leu, g/16 g of N 10.1 amino acids, DM, and N to values similar to (P 0.05) Lys, g/16 g of N 8.5 those obtained with birds fed the casein control supple- Total dispensable amino acids, g/16 g of N 48.6 mented with phytase. For many amino acids, digestibility Total indispensable amino acids, g/16 g of N 53.9 Total amino acids, g/16 g of N 102.5 values were returned to that of the casein control. In the Ca, g/kg 0.6 presence of 1 g of IP6 however, few nutrient digestibility Cu ND coefficients improved to the level of the casein control, KND Fe ND although the beneficial effect of phytase was still signifi- Mg, g/kg 0.1 cant; in the case of histidine and lysine, these values were Mn ND lower (P < 0.05) than for casein + phytase alone. Na, g/kg 12.6 P, g/kg 7.1 S, g/kg 5.0 Effect of IP6 and Phytase Zn, mg/kg 55.7 on the Excretion of Minerals 1Units are on an air-dry basis; ND = concentration below the detection limit of the inductively coupled plasma emission spectrometer. The effect of IP6 and phytase on the excretion of miner- als is presented in Table 3. No phytate-P was detected in the excreta from birds fed casein or casein with phytase. Chemical Composition of Casein The apparent digestibility coefficient of phytate-P was found to be low in the absence of exogenous phytase The determined chemical composition of casein is pre- (<0.2) and markedly higher (>0.7; P < 0.01) with the addi- sented in Table 1. Casein was found to have an N concen- tion of phytase. The true digestibility coefficient of sulfur tration of 138 g/kg (CP 862.5 g/kg = N × 6.25) and a reflected the digestibility coefficients of amino acids, with < DM content of 890 g/kg. The amino acids present in a decrease (P 0.05) associated with the ingestion of IP6 the highest concentrations in casein were glutamic acid, and an increase (P < 0.05) when the mixture of casein and leucine, lysine, aspartic acid, and valine. The mineral con- IP6 was supplemented with phytase. The presence of IP6 tent of casein was low, with sodium being present at the alone reduced the digestibility coefficient of sulfur by highest concentration (∼13 g/kg). The sulfur content of about 65%, whereas in the presence of phytase, the reduc- casein (5 g/kg) is assumed to be due to the presence of tion was only 26% compared with untreated casein. In sulfur amino acids and would thus yield a total sulfur the absence of supplemental phytase, the reduction in amino acid content of about 3 g/16 g of N, which is in the digestibility coefficient of sulfur associated with the agreement with published data (Degussa Corp., 1996). ingestion of IP6 (Table 3) was greater than the reduction in the digestibility coefficients of amino acids (Table 2). Effect of IP6 and Phytase on the The excretion of minerals was variably influenced by < Digestibility of DM, N, and Amino Acids the ingestion of IP6 and phytase. Increases (P 0.05) in the excretion of Ca, Mg, Mn, and Na by 187, 39, 87, and The effects of IP6 and phytase on the digestibility coef- 174%, respectively, by the ingestion of 1 g of IP6 were ficients of DM, N, and amino acids are presented in Table observed compared with birds fed casein alone. Supple- 2. Casein was found to be readily digestible, with true mentation of the mixture of1gofIP6 and casein with digestibility coefficients of DM, N, and total amino acids phytase reduced (P < 0.05) the excretion (percentage re- of 0.97, 0.85, and 1.01, respectively (Table 2). The addition duction compared with unsupplemented casein + 1 g of < of phytase to casein in the absence of IP6 decreased (P IP6) of endogenous Ca (51%) and Na (58%). The excretion 0.05) the digestibility coefficients of DM, N, and amino of P was increased by the ingestion of IP6, which can be acids compared with birds fed casein alone. There was a accounted for entirely by the excretion of P arising from < marked decrease (P 0.05) in the metabolizability of ca- IP6. The addition of phytase to the casein/IP6 mixture sein N (about 38%) by the addition of 2,000 FTU compared reduced (P < 0.05) the excretion of P compared with birds with the casein control. fed casein and IP6 without supplemental phytase, but The ingestion of 0.5 and 1 g of IP6 with casein reduced the excretion of P remained higher than in the birds fed (P < 0.05) the digestibility coefficients of casein compared casein alone. PHYTIC ACID, PHYTASE, AND PROTEIN UTILIZATION 881

Table 2. Effect of myo-inositol hexaphosphate (IP6) and phytase on the coefficient of true DM digestibility (TDMD), true metabolizability of N (TMN), and the true digestibility coefficients of amino acids of casein

1 Treatment (casein/IP6/phytase) 5 g/0 g/ 5 g/0 g/ 5 g/0 g/ 5 g/0.5 g/ 5 g/0.5 g/ 5 g/1 g/ 5 g/1 g/ 0 FTU 1,000 FTU 2,000 FTU 0 FTU 1,000 FTU 0 FTU 1,000 FTU SED

TDMD 0.97a 0.94ab 0.78b 0.50c 0.73b 0.37c 0.74b 0.08 TMN 0.85a 0.76ab 0.53bc 0.21cd 0.70ab 0.02d 0.54bc 0.12 Asp 1.01a 0.98ab 0.95ab 0.91bc 0.95ab 0.86c 0.92bc 0.04 Ser 1.02a 0.98a 0.96ab 0.89c 0.96ab 0.85c 0.90bc 0.03 Glu 1.01a 0.99ab 0.98bc 0.95cd 0.98bc 0.93d 0.96bc 0.02 Ala 1.04a 0.99ab 0.93ab 0.88bc 0.92ab 0.77c 0.87bc 0.06 Tyr 1.01a 0.99ab 0.98ab 0.95bc 0.97ab 0.92c 0.95bc 0.02 His 1.01a 1.00ab 0.97bc 0.95cd 0.97bc 0.92d 0.96c 0.02 Thr 1.02a 0.99a 0.97ab 0.87cd 0.96ab 0.81d 0.90bc 0.04 Arg 1.15a 1.10ab 1.08abc 1.00cd 1.01abc 0.96d 1.01bcd 0.05 Val 1.01a 0.98ab 0.97ab 0.94bc 0.97ab 0.91c 0.95bc 0.03 Phe 1.01a 0.98ab 0.97ab 0.94bc 0.96ab 0.91c 0.94bc 0.03 Ile 1.01a 0.98ab 0.97abc 0.94bc 0.96abc 0.92c 0.95bc 0.03 Leu 1.01a 0.98ab 0.97ab 0.94bc 0.96c 0.91ab 0.94bc 0.03 Lys 1.01a 1.00ab 0.97bc 0.96c 0.98bc 0.93d 0.96c 0.02 DIS2 1.01a 0.99ab 0.97abc 0.93cd 0.97abc 0.90d 0.94bcd 0.03 INDIS2 1.01a 0.98ab 0.96abc 0.92cd 0.96bc 0.88d 0.93cd 0.03 TAA2 1.01a 0.98a 0.97ab 0.92bc 0.96ab 0.89c 0.93bc 0.03 a–dValues in rows with no common superscript differ significantly (P < 0.05). 1 Weight of casein and IP6 fed in grams; units of phytase activity added in FTU; SED is the standard error of the difference. 2Mean of dispensable (DIS), indispensable (INDIS), and total amino acid (TAA) digestibility coefficients.

DISCUSSION may be a hypersecretion of digestive enzymes in a similar manner to the effects seen with protease inhibitors (Clarke The detrimental effect of IP6 on the utilization of N and Wiseman, 2003), potentially increasing endogenous and amino acids may be caused by several mechanisms. losses. Another possible mechanism is that IP6 may inter- Phytic acid may bind to or interact with dietary proteins, act with the , increasing enterocyte reducing their digestibility through changes in protein turnover, mucin secretion, or both (Cowieson et al., 2004). solubility or by altering the protein structure and thus Increased protein turnover, whether in the form of cells, reducing the activity of endogenous proteases because of mucin, or enzyme production, is likely to increase the loss steric hindrance. In addition, IP6 may bind to or interact of endogenous N and will certainly increase maintenance with endogenous enzymes and their cofactors, especially energy requirements. It is also feasible that IP6 increases polyvalent cations, thereby impairing their ability to di- catabolic processes within the animal such that N, in the gest dietary proteins. One consequence of such an effect form of , is excreted. If phytate disproportion-

Table 3. Effect of myo-inositol hexaphosphate (IP6) and phytase on the apparent digestibility coefficient of phytate-phosphorus (IPdig), true coefficient of sulfur digestibility (Sdig), and the excretion of minerals over 48 h from birds fed casein

1 Treatment (casein/IP6/phytase) 5 g/0 g/ 5 g/0 g/ 5 g/0 g/ 5 g/0.5 g/ 5 g/0.5 g/ 5 g/1 g/ 5 g/1 g/ 0 FTU 1,000 FTU 2,000 FTU 0 FTU 1,000 FTU 0 FTU 1,000 FTU SED IPdig — — — 0.10d 0.72b 0.19c 0.83a 0.03 Sdig 1.14a 1.10ab 0.73c 0.46d 0.87bc 0.34d 0.99ab 0.13 Ca, mg 31.5c 39.0bc 52.9b 52.9b 46.7bc 90.6a 44.7bc 12.7 Cu, mg 0.13 0.17 0.18 0.22 0.29 0.45 0.42 0.16 Fe, mg 1.89 2.07 1.87 3.03 2.79 6.11 4.32 1.92 Mg, mg 17.3c 18.3c 20.5bc 24.6a 19.5c 24.1ab 20.1bc 2.11 Mn, mg 0.23c 0.26bc 0.27bc 0.36ab 0.33abc 0.43a 0.38ab 0.06 Na, g 0.04b 0.03b 0.04b 0.05b 0.06b 0.10a 0.04b 0.01 P, g 0.16d 0.15d 0.20d 0.30bc 0.28c 0.48a 0.34b 0.03 K, g 0.16 0.19 0.18 0.15 0.20 0.15 0.18 0.01 S, mg 35.4d 36.3cd 45.7b 52.5a 42.2bc 55.6a 39.0b 3.35 Zn, mg 7.18 9.05 9.46 12.78 9.71 9.85 10.15 2.97 Total2 96.3a 101.7a 128.6a 147.4a 135.8a 236.0b 124.2a 15.31

a–dValues in rows with no common superscript differ significantly (P < 0.05). 1 Weight of casein and IP6 fed in grams; units of phytase activity added in FTU; SED is the standard error of the difference. 2Total excretion of minerals, excluding P and S (mg). 882 COWIESON ET AL. ately reduced the digestion, absorption, and utilization The extent of interaction between protein, minerals, of the most limiting amino acid, then there may be an and IP esters is directly correlated with the degree of increased intake of nitrogen in the form of the other amino phosphorylation of the inositol ring. Lower molecular acids. In the current study, the observation that sulfur weight IP esters such as mono- and diphosphates have digestibility was more severely depressed than that of any limited capacity to form chelates and to inhibit the digest- of the determined amino acids suggests that methionine + ibility of protein (Knuckles et al., 1989; Harland and Mor- cysteine may be affected to a greater extent by phytate ris, 1995; Harland and Narula, 1999). Furthermore, P from than the other amino acids. This may then increase the low molecular weight IP esters such as IP4 or IP3 has a metabolism of some of the other amino acids. relatively high availability for poultry and arguably, the Using an in vitro model, Knuckles et al. (1989) demon- esters do not constitute a major antinutritive problem, strated that inositol phosphate esters inhibited the diges- even for young chicks (Angel et al., 2002). Presumably, tion of casein by pepsin. The hexaphosphate (IP6) ester some of the beneficial effects noted in the current study used in the study by Knuckles and coworkers inhibited associated with the supplementation of casein and IP6 protein digestion by between 9 and 14%, which is in with phytase were due to dephosphorylation of IP6 into agreement with the effect of IP6 on the utilization of amino lower molecular weight IP esters with a reduced capacity acids in the present study. A reduction in true amino acid to interfere with amino acid utilization (Ravindran et digestibility coefficients by approximately 12% (Table 2) al., 2001). was noted with the addition of 1 g of IP6 to the casein Although the true digestibility coefficients of all amino suspension. In the in vitro model, the inhibition of protein acids were adversely affected by IP6, the amino acids digestion by inositol phosphate esters must be caused by affected to the greatest extent were alanine, serine, aspar- a direct reduction in the digestibility of protein, because tic acid, and threonine. These data support results from it cannot be explained by changes in the secretion of a previous study (Cowieson et al., 2004), which demon- endogenous amino acids. However, because IP6 impaired strated that the excretion of endogenous alanine, serine, protein digestion in vitro to a similar extent as in vivo aspartic acid, and threonine was increased by the inges- in the current study, it may be that the contribution of tion of IP by broilers. This suggests that some of the endogenous amino acids to the detrimental effects of IP 6 6 adverse effects associated with the ingestion of IP , and is relatively minor. However, the effect of IP on the loss 6 6 the amelioration of these with the addition of phytase in of endogenous N may be of greater consequence. the current study, can be explained by changes in the Phytate can form complexes between phytate, starch, secretion of endogenous amino acids. Endogenous secre- protein, and (by chelation) with many minerals, especially tions are rich in methionine, cysteine, threonine, and ser- divalent cations (Harland and Oberleas, 1999; Selle et ine (Mansoori and Acamovic, 1998a; Selle et al., 2000), al., 2000; Sandberg, 2002). The phytate anion has a net which supports the hypothesis that ingestion of IP stimu- negative charge of −3 at pH 1.5, increasing to −8ata 6 lates the secretion of endogenous compounds. This is pH of 7.5, the strong negative charge is caused by the further supported by the increased excretion of S in the dissociation of 8 protons from a total of 12 associated presence of IP indicating that the excretion of methionine with of inositol. These dissociating H ions 6 have pKa values of 1.5 to >8 (Costello et al., 1976; Harland and cysteine and minerals such as P, K, Na, and Ca, is and Oberleas, 1999). The negative charge of the phytate increased by the ingestion of IP6. Because the digestibility anion thus allows phytate-mineral-protein complexes to coefficient of S is likely to be directly related to the digest- form with mineral cations, low molecular weight carbo- ibility coefficients of the sulfur amino acids, the presence nium ions, and proteins at pH values lower than their of IP6 may have a significant effect on the requirement isoelectric points (Maenz et al., 1999; Selle et al., 2000). for sulfur amino acids. Because sulfur amino acids are The solubility of the phytate-mineral-protein complexes often the first limiting in young chick diets, IP6 may mark- is increased at low pH, such as gastric conditions, but is edly affect the protein efficiency ratio and net energy of decreased as pH increases, with maximum insolubility rations. Ravindran and coworkers reported that dietary at pH values between 4 and 7; that is, as found in the phytate concentrations were negatively correlated with distal gastrointestinal tract (Selle et al., 2000). Because inherent protein and amino acid digestibility, concluding casein contains relatively low concentrations of minerals that the adverse effects of phytate may be mediated compared with a standard broiler diet (Table 1), the for- through increased endogenous losses (Ravindran et al., mation of protein-phytate, rather than protein-phytate- 1999). Phytate interacts with the α-NH2 groups and side mineral complexes in the gastrointestinal tract is the more groups of basic amino acids (arginine, histidine, and ly- likely explanation for the effect of phytate on amino acid sine; Selle et al., 2000). It is interesting that, in the current digestibility. Increased proteolytic effects caused by phy- study, the digestibility coefficients of these amino acids tases may occur if the phytase reduces any antiprotease were amongst those that increased most by the addition of effects of phytate. However, the formation of protein- exogenous phytase (Table 2), which suggests that phytase phytate-mineral complexes may occur with minerals such reduces the interaction between IP6 and those amino as P, K, Na, and Ca from endogenous sources thus increas- acids. However, as mentioned previously, the extent to ing excretion and possibly reducing the availability of which endogenous losses contribute to the detrimental these ions as cofactors in enzymes. effects of IP6 in the current study remains unclear. PHYTIC ACID, PHYTASE, AND PROTEIN UTILIZATION 883

The adverse effect of phytase on the digestibility coef- salts of IP6 were not (Waldroup et al., 1964a,b). ficients of DM, the metabolizability of N and, to a lesser The interpretation given by those authors is based on extent, the digestibility coefficients of amino acid in the growth and tibia ash content and the presented informa- absence of phytase are interesting but difficult to explain. tion gives no indication of intake of diets and thus the The digestibility of amino acids is influenced to a small intake of P or of the relative digestibility coefficients of extent, but true metabolizable nitrogen and true dry mat- the various sources of P in the diets. Nonetheless, the ter digestibility in particular are dramatically reduced, source of inositol P and the conditions in which it is which suggests that phytase does not influence the inher- supplied, in a diet with active plant phytases and in the ent digestibility of the protein. Furthermore, it appears presence of divalent cations or in a system such as that that mucus or enzyme secretions are not increased be- used in this study, is highly likely to influence the uptake cause there is little effect on amino acid excretion. It ap- of dietary P from different sources. It is clear, however, pears that phytase reduces the utilization of absorbed from the current study that the presence of supplemental amino acids, causing an increase in the loss of nonprotein phytase increases the utilization of phytate-P, which was nitrogen and other DM components. It may be that in very low. Endogenous phytases in cereals can be present the absence of phytate, phytase stimulates catabolic path- at significant concentrations (>1,000 FTU/kg; Eeckhout ways increasing the loss of N. This may be related to the and De Paepe, 1994), although plant phytases have been timing and delivery of a balance of amino acids that is shown to be less effective than fungal or microbial phy- inappropriate, as suggested earlier. There may also be a tases, in degrading IP6 (Zimmermann et al., 2002). Fur- stimulation of an immunological response with a conse- thermore, some phosphatases require cofactors such as quential loss of biogenic amines. Degradation of endoge- calcium to function effectively (Oh et al., 2001) and it nous IP may also have occurred that may alter may be that as the birds in the present study were in an properties (Grases et al., 2001) or be required for normal abnormal nutritional state, the efficacy of endogenous metabolic or neurological systems. It has been shown phosphatases was low. The increase in the disappearance that endogenous inositol polyphosphate reserves play an of phytate-P associated with supplementation of IP6 with important role for supply of cell signaling molecules and exogenous phytase is supported by other studies in which maintenance of neurological health (Lees and Leong, similar responses have been noted (Rama Rao et al., 1999; 1996; Harland and Narula, 1999). Regardless of the mode Selle et al., 2000; Rutherfurd et al., 2002). It is likely that of action, a consistent decrease in nutrient digestibility the marked increase in the disappearance of phytate-P coefficients (Table 2) and an increase in the excretion of associated with the addition of phytase, compared with endogenous minerals (Table 3) were found with phytase unsupplemented treatments, is due to dephosphorylation addition to casein in the absence of IP6. However, these of the inositol ring and excretion of lower molecular adverse effects were not apparent when phytase was weight IP esters and free phosphate. The method used added to the IP6/casein suspension. Many phytases pref- in the current study to determine the concentration of erentially target higher molecular weight IP esters (Hu phytate-P in excreta detects all the esters of inositol phos- et al., 1996), dephosphorylating the inositol ring in a step- phate but does not detect free phosphate (Haug and wise manner starting either at the P in position 6 (in the Lantzsch, 1983). case of the phytase used in this study) or the P on carbon- Despite relatively high variation compared with the 3 in the ring (in the case of Aspergillus phytases; Kies, data presented in Table 2, the mineral excretion data pre- 1999). It is possible that the action of exogenous phytase sented in Table 3 demonstrate that the excretion of Ca, on IP esters, many of which are of relatively low molecu- Mg, Mn, and Na from endogenous sources was increased lar masses (Lees and Leong, 1996; Grases et al., 2001), by the ingestion of IP6. Esters of IP have the ability to was reduced when higher molecular weight IP esters chelate mineral ions and it is likely that this is not re- (added to casein as IP6) were available for hydrolysis. stricted to minerals present in feed but would also apply The apparent digestibility coefficient of phytate-P was to those secreted by the bird (Mansoori and Acamovic, between 0.10 and 0.19 without phytase supplementation 1998b; Lonnerdal, et al., 1999; Sandberg, 2002). The in- (Table 3) depending on the quantity of IP6 ingested by crease in the excretion of some endogenous minerals can the bird. These values are in agreement with results from be partially explained by the interaction between IP6 and other work, which demonstrated that in the absence of endogenous minerals, whereby chelation of minerals may exogenous phytase, the apparent digestibility coefficient cause them to be excreted. This removal of minerals may of phytate-P is between 0.10 and 0.40 and is dependent cause a negative feedback in which the animal secretes on the nature and type of feed provided (Ravindran et more minerals into the lumen of the gastrointestinal tract al., 1995; Miyazawa et al., 1996; Rutherfurd et al., 2002). in an attempt to maintain homeostasis. Thus, the effects The higher values obtained in some studies (>0.30) might of phytase and phytate are likely to have a greater effect be due to the presence of endogenous phytases in some on the performance of birds that are marginal in their animal feed ingredients that are not present in casein. supply of specific or in which the margin of Other workers have reported results that appear to con- the balance of dietary nutrients is small (Mansoori and tradict those found here in that supplementary P present Acamovic, 1998b; Lonnerdal et al., 1999). in IP6 and its sodium salt have been equally available The results reported here correspond well with those to young chicks as were inorganic phosphates, but that reported in a previous study (Cowieson et al., 2004), 884 COWIESON ET AL. where large increases in endogenous Na excretion were Eeckhout, W., and M. De Paepe. 1994. Total phosphorus, phy- noted when 1 g of IP was fed to feed-deprived birds. It tate-phosphorus and phytase activity in plant feedstuffs. 6 Anim. Feed Sci. Technol. 47:19–29. may be that the entry of acid into the duodenum stimu- Ferraz de Oliveira, M. I., K. Hillman, and T. Acamovic. 1994. lated an increase in the secretion of bicarbonate and bile The effects of enzyme treated and untreated lupins and their salts in an attempt to maintain duodenal pH, increasing alkaloids on poultry gut microflora. Pages 195–200 in Plant- the measured excretion of some mineral ions, possibly associated toxins: Agricultural, and ecological including sodium. aspects. S. M. Colgate and P. R. Dorling, ed. CAB Publ., Wallingford, UK. In conclusion, the reduced digestibility coefficients and Grases, F., B. M. Simonet, R. M. Prieto, and J. G. March. 2001. increased excretion of minerals in birds fed mixtures of Variation of InsP4, InsP5 and InsP6 levels in tissues and casein and IP6 presented here correspond well with those biological fluids depending on dietary phytate. J. Nutr. 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